ABSTRACT Leptospirosis is considered by the Centers for Disease Control to be the most widespread zoonosis in the world. Reservoir hosts with chronic renal tubular infection typically transmit pathogenic Leptospira species to humans through urinary shedding. Leptospiral infection in humans frequently results in fulminant liver dysfunction, kidney failure, and severe pulmonary hemorrhage syndrome with a mortality rate of >10%. Leptospirosis has emerged as a major public health burden in urban slums where risk is strongly linked to poverty and rat exposure. L. interrogans is the most common organism isolated from the urban brown rat and is also the predominant cause of human leptospirosis in urban slums. The overall goal of this proposal is to identify leptospiral virulence proteins and understand their role(s) in pathogenesis. Given that leptospiral pathogens both persist in the ambient environment and colonize host tissues as essential parts of their life cycle, it is not surprising that their genomes are richly endowed with at least 152 different signal transduction proteins. High throughput massively parallel transposon sequencing resulted in identification of several novel virulence genes including LIC12327, a putative adenylate cyclase. Further, we have discovered that LIC11484, a unique leptospiral transcriptional regulator with a cyclic nucleotide binding domain and helix-turn-helix DNA binding domain, is required for virulence. These data have led to the hypothesis that cAMP is a central virulence messenger of L. interrogans. We propose the following two Aims: 1) What genes are required for leptospiral virulence? We will screen a transposon mutant library for mutants with in vivo fitness defects by high throughput massively parallel transposon sequencing. We will confirm virulence defects of mutants and complemented strains by qPCR and LD50 studies. Identification of such genes will facilitate a greater understanding of leptospiral virulence. 2) Are cyclic nucleotides involved in leptospiral virulence regulation? We will determine whether LIC12327 is an adenylate cyclase using E. coli reporter strains that respond selectively to either cAMP or cAMP and cGMP. We will measure cAMP and/or cGMP in L. interrogans and examine how cyclic nucleotide levels change in response to in vivo-like conditions such as physiologic osmolarity and temperatures ranging from ambient to 37C. We will measure the affinity of LIC11484 for cyclic nucleotides. RNAseq will be performed with wildtype L. interrogans, LIC12327 mutants, and LIC11484 mutants to identify genes potentially regulated by cAMP and/or LIC11484.